COMPOUNDS USEFUL IN ANTI-ALLERGY TREATMENT

Abstract: Pharmaceutically acceptable compounds of less than 1200 MW for use in the treatment of IgE-mediated allergies comprising a first negatively charged atom or group and a second negatively charged atom or group, separated by a spacing group effective conformationally to position said negatively charged atoms or groups so that they will neutralize the lysine residues of the amino acid sequence Lys Thr Lys at positions 497-499 of the C ELEMENT 4 constant domain of cell-bound human IgE, but excluding any compounds already known for this purpose such as DSCG and nedocromil sodium and toxic compounds. In a second aspect, the present invention provides peptides of formula (I) (in which the left-hand side represents the N-terminus and the right-hand side the C terminus): R<1>m Xaa<1> Sp Xaa<2> R<2>n wherein: R<1> represents a residue of an amino acid or of a sequence of two or three amino acids, which may be the same or different; R<2> represents a residue of an amino acid or of a sequence of two or three amino acids, which may be the same or different; (but preferably any amino acid residue of R<1> or R<2> adjacent to an Xaa residue is neither positively nor negatively charged); Xaa<1> represents a residue of a negatively charged amino acid, preferably Glu; Sp represents a spacing residue, preferably of a non-charged amino acid, preferably Pro, or of a non-charged dipeptide, which provides the spacing required for the negatively charged groups of the Xaa residues to be sufficiently proximal to the lysine residues of the amino acid sequence Lys Thr Lys at positions 497-499 of the C ELEMENT 4 constant domain of cell-bound human IgE to neutralise them; Xaa<2> represents a residue of a negatively charged amino acid, preferably Glu; and m and n denote the number of amino acids in R<1> and R<2> respectively and each of m and n independently is 0, 1, 2 or 3, and the sum of m plus n is from 0 to 3; and their terminal functional derivatives; both per se and for use in the treatment of IgE-mediated allergies.

Description: COMPOUNDS USEFUL IN ANTI-ALLERGY TREATMENT Background of the invention 1. Field of the invention The invention relates to compounds for use in anti-allergy treatment and, particularly, to a class of peptides useful in treating IgE-mediated allergies. 2. Description of the related art Allergic symptoms are brought about through the release of vasoactive amines (mediators) , notably histamine, from cells into the surrounding tissue and vascular structures. Histamine is normally stored in special cells known as mast cells and basophil leucocytes. The mast cells are dispersed throughout animal tissue whilst the basophils circulate within the vascular system. Thase cells manufacture and store histamine within the cell unless a specialised sequences of events occurs to trigger its release. The role of immunoglobulin E (IgE) antibodies in mediating allergic reactions is well known. IgE is a complex arrangement of polypeptide chains which, as in other immunoglobulins consists of two light and two heavy chains linked together by disulphide bonds in a "Y" shaped configuration. Each light chain has two domains, one variable (V L ) domain linked to a domain with a relatively invariant amino acid sequence termed a constant domain ( C L ) . Heavy chains, by contrast, have one variable domain (V H ) and in case of IgE, four constant domains (CH lf CH 2 , CH-., CH 4 , also known as Cel, Ce2, Ce3, Ce4) . The two "arms" of the antibody are responsible for antigen binding, having regions where the polypeptide structure varies, and are termed Fab' fragments (fragment - antigen - binding) or F(ab')2 which represents two Fab' arms linked together by disulphide bonds. The "tail" or central axis of the antibody contains a fixed or constant sequence of peptides and is termed the Fc fragment (fragment - crystalline) . The Fc fragment contains the antibody's biologically active sites which enable the antibody to communicate with other immune system molecules or cells by binding to their Fc receptors . Fc receptors are molecules which bind with high affinity and specificity to molecular active sites within immunoglobulin Fc regions. Fc receptors may exist as integral membrane proteins within a cell's outer plasma membrane or may exist as free "soluble" molecules which freely circulate in blood plasma or other body fluids . Figure 1 of the drawings shows the structure of an antibody molecule and the location of the antigen binding sites (Fab' arms) , the Fc fragment, and the active sites which includes the cell binding site. Active sites, depending on their function, may already be exposed and therefore able to bind to cellular receptors . Alternatively, they may be hidden until the antibody binds to the antigen, whereupon the antibody may change in structure and subsequently expose other active sites which can then trigger a specific,.,immune activity. The allergic (immunologic) release of histamine within the organism from the mast cells and basophils can only occur under the following circumstances. An IgE molecule must lock onto or attach itself at its Fc end to the cellular Fc receptor site, thus securing the IgE molecule to the mast cell or basophil (Figure 2a) . The Fab' portions of the cell-bound IgE molecules must be cross-linked by a particular compatible antigen (the allergen) . Should such an interaction occur (Figure 2b) , the mast cell or basophil is automatically triggered to release histamine to the local environment, manifesting familiar allergic symptoms. Conventional approaches to allergy treatment have involved systemic therapy with anti-histamines or attempts to desensitise patients, approaches with have not addressed themselves to the basic IgE-mast cεll/basophil interaction. Other prior art has concerned itself with the production of polypeptide chains capable of blocking the binding of the IgE antibody to the Fc receptors on the cell surfaces and displacing IgE from binding sites upon which IgE is already bound (Figure 3) . In PCT Patent Application WO 90/15878 (Stanworth, et al) it has been suggested that a certain part of one of the constant domains of human IgE, namely amino acid residues 497-506 of the Ce4 chain, acts as an effector of histamine release, but only when the IgE has undergone a postulated change in conformation as a result of interaction with the allergen. Assuming the validity of the "second receptor" hypothesis, it is not clear how to prevent the interaction between the postulated "effector site" on the anaphylactic (IgE) antibody molecule and this "second receptor" on the cell surface. Presumably the cross linking of mast cell bound IgE antibody molecules by a specific antigen (allergen) induces a conformational change within their Fc regions bringing the "effector -site" into close juxtaposition with the "second receptor". In this event, any attempt to block this interaction could encounter problems of steric hindrance in the region of the supposed effector site. The patent application proposes an anti-allergy treatment based on in vivo stimulation of production of antibodies to a peptide of sequence which is identical with or similar to that of the "497-506" effector portion of the IgE. This effector sequence (SEQ ID NO: 1) is as follows: Lys Thr Lys Gly Ser Gly Phe Phe Val Phe 1 5 10 The effector peptide, which has SEQ ID NO: 1, is administered in some suitably "non-self" form, e.g. as a conjugate to a protein. Antibodies and polyclonal antiserum to this peptide have also been shown to inhibit IgE-mediated allergic reactions . With the exception of Stanworth, generally the prior art has concerned itself with the development of "blocking peptides" for the prevention of the binding of IgE to mast cells and basophils. The development of "anti-binding site antibodies", has elucidated the Fc binding site and subsequently, blocking anti-peptides were developed [Burg, E. et al, Molecular Immunology, 24., pp. 379-389 (1987)] . Hahn (WO 86/01211: Merck Patent GmbH) suggested that immunotherapeutic agents might be found amongst a class of compounds which block immune complex binding to immunoglobulin Fc receptors and/or which block immunoglobulin binding to lymphocyte Fc receptors. Hahn's reason for looking at putative Fc blocking peptides is stated as follows: "It is only after antibodies bind to antigen or are otherwise caused to aggregate that active sites within the Fc region are able to bind to and activate Fc receptors . Fc receptors are, therefore, the critical link between antibodies and the remainder of the immune system. Fc receptor.binding to antibody Fc region active sites may thus be characterized as the "final common pathway" by which antibody functions are mediated. If an antigen-bound antibody does not bind to an Fc receptor, the antibody is unable to activate the other portions of the immune system and is therefore rendered functionally inactive. Any peptide with the ability to bind to immunoglobulin Fc receptors has therapeutic usefulness as an immunoregulator by virtue of the peptide' s ability to regulate binding to the receptor. Such an Fc receptor "blocker" occupies the immunoglobulin-binding site of the Fc receptor and thus "short circuits" the immunoglobulin' s activating ability". Hahn lists many thousands of compounds in the patent application, all of which are alleged to be active site compounds which block immune complex binding to Fc receptors . However, there is no teaching of any theory or principle of selection of effective Fc binding "blocker" compounds. It is known that the IgE antibody is often firmly bound to the mast cell or basophil [Stanworth, D.R., Nature 233 , pp. 310-316 (1971)] , even without the presence of an allergen, and it is only when the allergen is present that the supposed "second receptor" is triggered and the histamine is released. Thus, following the teaching of Hahn, merely blocking the site at which IgE binds to the mast cells would inhibit IgE function of only those IgE molecules which freely circulate and are not yet attached to the mast cells or basophils. This approach would be unsuitable where cell-bound IgE is already present, unless such a blocking peptide is also able to displace already bound IgE. This approach was taken by Hamburger who reported that a pentapeptide from the C H 2 domain of human .IgE was capable of competing with IgE specific binding sites on mast cells in human skin [Hamburger, R.N. , Science 189, pp. 389-390 (1975)] . This result could not however be confirmed by ot:ιer investigators [Bennich, H.H., et al, Int. Arch. Allergy Appl. Immunol., 53 , pp. 459-468 (1977)] . It is to be noted that a patient who has already been exposed to allergen will have allergen-specific cell-bound.,IgE. The leading anti-allergy drugs are disodium cromoglycate (DSCG) and nedocromil sodium, marketed by Fisons PLC as "Intal" and "Tilade". It is desirable to find alternatives to these drugs for the benefit of patients who do not tolerate them well and because their insolubility limits the methods by which they can be administered, e.g. by inhaler or aerosol respectively. Summary of the invention It has now surprisingly been found that certain short peptides show anti-allergic activity when administered as they are, i.e. without any attempt to raise antibodies to them. These peptides comprise two negatively charged amino acid residues, especially of glutamic acid (Glu) , separated by an uncharged amino acid such as proline (Pro) or glycine (Gly) . Three-dimensional models suggest strongly that the mode of action of these peptides is to bind to a part of the effector peptide, SSQ ID NO: 1, namely to the Lys Thr Lys region. The evidence of modelling suggests strongly that negative charges on the peptides of the invention bind strongly to the doubly charged Lys Thr Lys "head" of the effector site, thus neutralising its effector activity and therefore the release of histamine triggered by the action of the IgE on mast cells. A further constraint is a requirement for the residue or residues spacing the negatively charged groups of the compound according to the invention to react via non-covalent forces with the Thr residue separating the key Lys residues of the peptide SEQ ID NO: 1 (the effector or "trigger" peptide) . From these considerations, it follows that other small compounds, which are not peptides, but which have negatively charged groups or atoms spaced apart by about the same distance as those in the preferred tripeptide Glu Pro Glu, will act in the same way to combat IgE-mediated allergies. These considerations are made even more credible by the fact that the marketed drugs DSCG and nedocromil sodium have negative charges spaced by l£ and 10 Angstrom units (1.5 and 1.0 nm) respectively and that these separations are similar to those in the said tripeptide. In a first aspect, therefore, the present invention provides small compounds for use in the treatment of IgE-mediated allergies comprising a first negatively charged atom or group and a second negatively charged atom or group, separated by a spacing group effective conformationally to position said negatively charged atoms or groups so that they will neutralize the lysine residues of the amino acid sequence Lys Thr Lys at positions 497-499 of the Ce4 constant domain of cell-bound human IgE, but excluding any compounds already known for this purpose such as DSCG and nedocromil sodium and toxic compounds . For the purposes of this application the term "small" is defined as a molecular weight (MW) less than 1200. In a second aspect, the present invention provides short peptides of the following formula I (in which the left-hand side represents the N-terminus and the right-hand side the C- terminus) : R Xaa 1 Sp Xaa 2 R 2 n (Formula I) wherein: R 1 represents a residue of an amino acid or of a sequence of two or three amino acids, which may be the same or different; R 2 represents a residue of an amino acid or of a sequence of two or three amino acids, which may be the same or different; (but preferably any amino acid residu- ■" R 1 or R 2 adjacent to an Xaa residue is neither positively negatively charged) ; Xaa 1 represents a residue of a negativ:-_y charged amino acid, preferably Glu; Sp represents a spacing residue, preferably of a noncharged amino acid, preferably Pro, or of a non-charged dipeptide, which provides the spacing re uired for the neftively charged groups of the Xaa residues to be sufficiently £ roximal to the lysine residues of the amino acid sequence Lys Thr Lys at positions 497-499 of -the Ce4 constant domain of cell-bound human IgE to neutralise them; Xaa 2 represents a residue of a negatively charged amino acid, preferably Glu; and m and n denote the number of amino acids in R 1 and R 2 respectively and each of m and n independently is 1, 2 or and the sum of m plus n is from 0 *- ■ -- 3; and their terminal functional deri" ves . For t. rposes -o - applicati' he term "short" i fined as 6 ass amino 3 (aa) . nvention includes such peptides both per se and for use in i -py- especially for treating IgE-mediated allergies. Where legally permissible, the invention includes a method of treating a patient having symptoms of IgE-mediated allergy, which comprises administer-*ng to ε pa. ent a therapeutically effective amount of mpounσ defined above, especially a p -ntide as defined ; r 2r aspects of present invention are defined in the claims . The term "terminal functional derivative" as used herein includes any derivative formed by reaction of the terminal amino or carboxy group and which does not alter adversely the character imparted to the peptide by the other amino acid residues. Thus, any conventional derivative, especially N- acetyl and/or C-amide, can be used in this invention. Description of the preferred embodiments The peptides used in this invention have a length of from 3 to 6 amino acid residues (or spacing equivalent when Sp is not an amino acid or dipeptide residue) . The essential feature of the peptides used in this invention is that they should contain two negatively charged amino acids spaced apart by an appropriate spacer group. The spacer group should perform the function of spacing the negative charges on the negatively charged amino acids, so that they lie adjacent to the positive charges on the lysines of the IgE chain referred to above. The positive charge in lysine lies on the amino group nitrogen atom of a 4-aminobutyl side chain. Computer modelling by the inventors predicts that these nitrogens lie about 1.4 nm apart and that the negative charges in DSCG and nedocromil sodium are about 1.5 and 1.0 nm apart respectively, suggesting that these distances are about the maximum and minimum spacings for neutralisation of the lysines. In the preferred tripeptide Glu Pro Glu, the spacing between the negatively charged oxygens in the 2-carboxyethyl side-chains of glutamate is about 1.3 nm. Here, the spacing group is the single amino acid residue proline. If another amino acid were included in the spacing group, i.e. if it were a dipeptide, the spacing between the negative charges on the glutamates would increase by about the length of two C-N bonds, roughly 0.3 nm, to 1.6 nm. which is a little too great for the best neutralising effect. However, if the negatively charged amino acids were aspartates, which have shorter, carboxymethyl, side chains, the spacing group would have to be made correspondingly longer, i.e. usually a dipeptide residue. It would be possible for the peptides to have different negatively charged residues, such as one glutamate and one aspartate, but this particular combination may give rise to some spacing problems as the single amino acid spacer is a little short, while the dipeptide spacer is a little long, although it is likely that the effects of the invention would be obtained in some degree with either. Using these guidelines, the person of average skill in the art will be able to obtain the benefits of this invention for a variety of different peptides. The amino acids herein can have the (D) or (L) configuration, but are preferably selected from the 20 naturally occurring (L-) amino acids. Thus, the preferred negatively charged amino acids are aspartic (Asp) or glutamic (Glu) . The spacing group Sp preferably comprises (consists of when it is a single amino acid residue or includes when it is a dipeptide residue) proline (Pro) or a ring-substituted derivative thereof. *.« The peptides are thought to bind to the patient's IgE, after the IgE has supposedly undergone a conformational change as a consequence of interaction with the allergen, thereby blocking its subsequent action of triggering mast cells. Such triggering initiates release of histamine. Therefore, it is considered essential to the invention that the two negatively charged amino acid residues, appropriately spaced apart, should be presented to the IgE. Accordingly, the peptide should not contain any amino acids which are likely to interfere with such presentation or cause the peptide to bind to other proteins. Thus, the spacing group preferably does not comprise cysteine, which might cause binding to other proteins. The peptide need not be any longer than a tripeptide, as in preferred peptides Glu Pro Glu and Glu Gly Glu, but it can have additional, flanking amino acid(s) at either or both sides of the essential motif Xaa 1 Sp Xaa 2 . The nature of any flanking amino acid or peptide chain represented by R 1 or R 2 is not critical to the invention, although an amino acid externally adjacent to either of the negatively charged amino acids, i.e. to the N-terminus of Xaa 1 or to the C-terminus of Xaa 2 is preferably not negatively charged, especially not Glu or Asp, or positively charged, especially not Lys, Arg or His. From the viewpoint of ease of synthesis, it is preferred that any flanking amino acids be the same within each flank or both flanks, e.g. as in another peptide of the invention, having the sequence (SEQ, ID NO: 2) : Ala Ala Ala Glu Pro Glu 1 5 The peptides of the invention may be prepared by standard methods, e.g. the well-known Fmoc. method. The peptides used in the invention are preferably administered as such, i.e. without any carrier such as a conjugated protein or a branched peptide type of carrier, as it is not desirable that the patient should raise antibodies against them. It may well be desirable, however, that they be administered in a slow release or depot form, for example in liposomes or nanoparticles (isobutyl and isodecylcyanoacrylate carriers used for anti- cancer drugs) . The peptides used in the invention may be administered by any of the conventional routes of giving drugs, for example orally, by intravenous or intramuscular injection or as eyedrops or as a nasal spray. Pharmaceutical compositions of the invention comprise a peptide of the invention and a carrier or diluent. Oral preparations include solid forms such as tablets, pills, lozenges and capsules, and liquid forms such as solutions in containers and sprays, e.g. nasal and throat sprays, all of which can be formulated in any conventional way. Injectable preparations include sterile solutions, especially in physiological saline. As the peptides are readily soluble, they can be administered very easily in a liquid form. It will usually be desirable to give the peptide in several doses over an extended period, each dose typically containing from 0.01 to 1 mg. oi che peptide. The peptide is best given prophyi tically, so as to be available to block the IgE effectc- site immediately the allergen binds to the IgE, but the invention also includes therapeutic treatment. Turning now to the non-peptide compounds for use in treating allergies, the compounds should aim to mimic the tripeptide Glu Pro Glu structurally a- "herefore one class of preferred compounds has the gener. .rmula (II) : MOOC-CH,-CH 2 -Y-CH 2 -CH 2 -COOM (II) wherein M represents a hydrogen atom or a non-toxic cation such as sodium or potassium and Y represents a group which spaces the two 2-carboxyethyl groups apart so that the negative charges (when in anionic form) are separated by a distance of from about 1.0 to less ? than 1.6 nm. Roughly stated, the length of Y is about equal to six saturated C-C or C-M bond lengths, about 0.15 x 6 = 0.9 nm. The Y residue could simply be an aliphatic chain, as in a 1, 5pentylenediamine N,N-di- (3-propionεte) , but preferably has some rigidity to mimic the planar carbonyl groups in peptide linkages and possibly additional rigidity as conferred by the pyrrolidine ring in the preferred proline spacer amino acid. This may be provided by at least one carbocyclic or heterocyclic ring within Y, as in 1,2- cyclopentylene and 1, 2-pyrrolidinylene compounds or in bicyclic ring systems. The compounds must, of course, be non-toxic and stable to rapid metabolic degradation. They may be organic or organometallic in nature . The formulation and administration of the non-peptide compounds are in principle no different from that of the peptides, as described above, but variations and limitations may be necessary according to the type of compound chosen. In particular, the dosage may need to be higher, e.g. dosages of 1 to 100 mM may be required. The following Examples illustrate the invention. EXAMPLE 1 This Example relates to the testing of the peptide Glu Pro Glu for anti-allergy activity. Part A: In vitro rat mast cell test Rats were experimentally sensitised by injection with allergen (ovalbumin) and Bordetella pertussis, employing a regimen previously described by D.R.Stanworth et al . , The Lancet. 336, 1279-1281 (1990) . Their peritoneal mast cells were isolated by washing out the peritoneum twice with cold (4 deg. C.) calcium ion-free HEPES-buffered Tyrode salt solution (HBT) , pH 7.2. (HBT contains 13,.7 mm sodium chloride; 2.7 mM potassium chloride; 0.64 mM sodium dihydrogen phosphate dihydrate; 0.05 mM magnesium chloride hexahydrate; 0.1 mM calcium chloride dihydrate; 1 mM HEPES; 0.56 mM D-glucose and 1 mg./ml. gelatin) . The resulting cell suspension, at 4 deg. C. , was centrifuged for 5 minutes at 2000 r.p.m. , the supernatant was discarded and the solid residue re-washed with the cold buffer. This process was repeated three times. If the supernatant was not clear at the end of the final wash, the cells were discarded. The cells were finally re- suspended in HBT buffer, this time containing 0.1 mM calcium ions, provided as the chloride. The cells were then recentrifuged and resuspended in the calcium ion-containing HBT buffer at a concentration of about 20,000 cells per 100 microlitres in 1.5 ml. tubes. The mast cells were incubated with different concentrations of the peptide under test for 15 minutes at 37 deg. C. An allergen to which rats had been experimentally sensitised, namely ovalbumin, was then added, as a 0.5 mg./ml. solution in calcium ion-containing HBT buffer and the cells were then incubated for a further 15 minutes at the same temperature. At the end of the period of incubation, 1 ml . of cold calcium ion-free HBT buffer was added to each sample to quench the reaction and the tubes were then centrifuged at low speed in an MSE Microfuge centrifuge for 1 minute. The supernatants were removed by fine-tipped pipettes and added to tubes containing 1 ml . of 0.8M perchloric acid. The tubes were then centrifuged at 2,000 r.p.m. for 5 minutes and stored at 4 deg. C. overnight or at -20 deg. C. over longer periods. 1 ml . of 0.4M perchloric acid was added to the pellet. The tube was then whirled for 20 seconds to mix the contents and sonicated for 5 minutes to thoroughly lyse the cells. The cells were then centrifuged in the MSE Microfuge at high speed for 1 minute. The cell pellets thus obtained were stored as described above for the supernatants . The percentage of histamine recovered from the mast cell supernatants and lys--d pellets by solvent extraction was then reacted with ortho-phthalaldehyde to produce a fluorophore and measured us-i,ng a spectrafluorimetric assay, as first described by D.P.Evans et al. , Life Sciences 12., Part II, 327-336 (1973) . The amounts of histamine in the supernatants, i.e. released by the samples incubated with the peptide, were expressed as a percentage of the total histamine available (from supernatants plus cell pellets) and a percentage inhibition was determined by reference to the amount of histamine released by a control sample to which buffer was added without any peptide. The results are shown in Table 1 below. All results are the mean of duplicates. Table 1: Inhibition of histamine release from actively sensitised rat peritoneal mast cells Cone, of test Histamine released Inhibition peptide (mM) (as % total available) (as % of control) 3.20 12.0 45.9 1.60 11.3 49.1 0.80 12.6 43.2 0.40 11.2 49.8 0.20 12.8 42.3 0.10 13.4 39.6 0.05 13.1 41.0 Control 22.2 0.0 (buffer only) Part B: Repeat of in vitro rat mast cell test, with the addition of a comparison with nedocromil sodium The test of Part A was repeated, but using different concentrations of the peptide and also performing runs in which the cells were incubated with various concentrations of the known drug nedocromil sodium. The results, which are the mean of duplicates, are shown in Table 2 below. Table 2 : Inhibition of histamine release from actively sensitised rat peritoneal mast cells Cone, of test Histamine released Inhibition compound (mM) (as % total available] (as % of control) Glu Pro Glu 1.300 27.2 22.1 0.540 23.6 32.4 0.270 25.6 26.7 0.135 22.4 35.8 0.027 21.0 39.8 0.014 30.1 13.7 0.003 33.6 3.7 Nedocromil sodium 2.50 18.4 47.3 1.25 21.6 38.0 0.25 23.5 32.7 Control 34.9 0.0 (buffer only) EXAMPLE This Example relates to the testing of the peptide Glu Pro Glu with various other substances for comparison, by the method of Example 1. The results shown in Table 3 below are the mean of duplicates. Table 3 : Inhibition of histamine release in actively sensitised rat peritoneal mast cells Peptide % inhibition of histamine release under at peptide concentrations, mM test 3.0 1.0 0.33 0.17 Glu Pro Glu 8.2 28.7 30.3 23.8 Comparative % inhibition of agents histamine release Murine monoclonal antibody to peptide SEQ ID NO: 1 30.3 (diluted 1:100) Rat antiserum to peptide SEQ ID NO: 1 (undiluted) 23.8 Nedocromil sodium (0.25 mg./ml. = 0.6mM) 23.8 It will be seen that the preferred Glu Pro Glu peptide of the invention gave at least as good a result as nedocromil sodium and the antisera to the peptide of SEQ ID NO:l mentioned above. EXAMPLE 3 This Example relates to the testing of several peptides of the invention by a different method, which depends on the inhibition of allergen-induced histamine release from passively sensitised human lung mast cells. A piece of normal human lung tissue (about 7 g.) was stored at 4 deg. C. in calcium ion-free HEPES-buffered Tyrode (CFHBT) solution until use. The piece of lung was washed three times with CFHBT to remove any blood cells and blood clots. It was then cut into 1 cm. x 2 cm. pieces, which were then chopped in a Mcllwain tissue chopper, making five passages of the blade and employing 2 micrometre cuts on high-speed and medium blade force. The resulting chopped tissue was pooled and aliquots placed into a 50 ml. tube containing cold (4 deg. C.) CFHBT. The pooled chopped ti-gsue, which provided the lung mast cells, was then washed with the cold CFHBT and the cells thoroughly dispersed with a plastic Pasteur pipette. The dispersion was centrifuged at 2000 r.p.m. for 5 minutes at 4 deg. C. The supernatants were discarded and fresh cold CFHBT added. This washing was repeated thrice. If the supernatant was not clear by the end of the final wash, the cells were discarded. Allergic serum from a rabbitsensitive individual ("IVL") was added to washed chopped human lung (1 ml. serum/g. wet weight of cells; 1 g. cells/5 ml. CFHBT) . The cells were then mixed for 18 hours at room temperature in a Denley Spiramix (which keeps them continuously suspended as a result of its gentle rolling/rotating motion) . The cells were finally made up in HBT at 4 deg. C, using 100 microlitres of cells (about 50 mg. per 1.5 ml. tube) . It will be seen from Table 4 that in this test the peptides of the invention gave the same or better results than the marketed drug nedocromil sodium. While N-acetylation or Camidation individually did not seem to affect the inhibition of histamine markedly, there was a reduction in the inhibition when both termini were blocked in these ways . Table 4 : Inhibition of histamine release from passively sensitised human lung-mast cells Peptides % inhibition of histamine release under at peptide concentrations, mM test 2.0 0.2 0.02 0.002 0.0002 0.00002 Glu Pro Glu 53.7 40.6 51.4 20.0 13.1 15.4 Glu Gly Glu 8.0 10.3 Ac-Glu Pro Glu 45.1 45.1 17.7 Glu Pro Glu-NH, 39.4 22.9 Ac-Glu Pro Glu-NH, 22.9 6.9 17.1 Comparative, agent Nedocromil sodium 4.0 29.7 11.4 EXAMPLE 4 This example relates to the testing of 24 peptides according to the invention in a comparison against the known compound Nedocromil Sodium. Table 5 lists the tested compounds and shows the charge spacing in Angstrom units A. Standard single letter codes for amino acids are used throughout the Table. The test uses an ELISA screen to determine the concentration (mM) of compound required to achieve a set level (25% or 50%) of inhibition of the peptide SEQ ID NO:l (the "effector" peptide for histamine release) . It can be seen from the results that the peptides of the invention as shown have effectiveness comparable to that of the known compound nedocromil sodium, and in some cases the peptides are more effective (i.e. lower dosage required for the same level of inhibition) . Table 5 : Compound Charge Inhib. Cone. (mM) Spacing A 25% 50% Nedocromil 12.1 0.25 1.25 Sodium F229 AcEPE 12 0.16 2.7 F237 EGFGE 10.6 0.5 >2 F239 EGFE 14.1 0.26 0.15 F241 EGFK 12.3 0.5 1 F2 2 AcEGKE 12 0.4 1.6 F250 EKE 10.2 0.15 0.3 F252 AcEKE 10.2 0.3 >2 F241 AcEGFK 12.3 0.2 0.5 F288 DAAPD 12.8 0.6 0.1 F303 EPPE., 14.6 0.4 0.8 F249 EFE 10.2 0.3 1.2 F285 DAPD 11.8 0.15 1.2 F2 1 DAAAPD 12 0.6 0.2 F254 AEPEA 12.2 0.6 0.5 F236 EEKGE 12.8 1 1.3 F255 EPEA 13.2 0.4 1.1 F259 AEPE 12.1 0.3 1.1 F248 ACEKGE 12.8 0.3 0.8 F251 AcEFE 10.2 0.3 1.2 F228 EGE 13 0.4 0.7 F230 AcEGE 13 0.1 1.5 F235 EGFGE 13.5 0.2 0.5 F78 EPE 12 0.3 1.3 F237 ACEGFGE 13.5 0.5 0.7 The following claims define some important features of the invention but should not be construed as detracting from the generality of the inventive concepts hereinbefore indicated. 23 Claims 1. Pharmaceutically acceptable compounds of less than 1200 MW for use in the treatment of IgE-mediated allergies comprising a first negatively charged atom or group and a second negatively charged atom or group, separated by a spacing group effective conformationally to position said negatively charged atoms or groups so that they will neutralize the lysine residues of the amino acid sequence Lys Thr Lys at positions 497-499 of the Ce4 constant domain of cell-bound human IgE, but excluding any compounds already known for this purpose such as DSCG and nedocromil sodium and toxic compounds. 2. Compounds according to claim 1, wherein the negative charges are separated by a distance of at least 1.0 but less than 1.6 nm. 3. Peptides of the following formula I (in which the left-hand side represents the N-terminus and the right-hand side the C- terminus) : R\. Xaa 1 Sp Xaa 2 R 2 n (Formula I) wherein: R 1 represents a residue of an amino acid or of a sequence of two or three amino acids, which may be the same or different; R 2 represents a residue of an amino acid or of a sequence of two or three amino acids, which may be the same or different; (but preferably any amino acid residue of R 1 or R 2 adjacent to an Xaa residue is neither positively nor negatively charged) ; Xaa 1 represents a residue of a negatively charged amino acid, preferably Glu; Sp represents a spacing residue, preferably of a noncharged amino acid, preferably Pro, or of a non-charged dipeptide, which provides the spacing required for the negatively charged groups of the Xaa residues to be sufficiently proximal to the lysine residues of the amino acid sequence Lys Thr Lys at positions 24 497-499 of the Ce4 constant domain of cell-bound human IgE to neutralise them; Xaa 2 represents a residue of a negatively charged amino acid, preferably Glu; and m and n denote the number of amino acids in R 1 and R 2 respectively and each of m and n independently is 0, 1, 2 or 3 , and the sum of m plus n is from 0 to 3 ; and their terminal functional derivatives. 4. Peptides according to claim 3, wherein the charges on the negatively charged amino acids Xaa 1 and Xaa 2 are separated by more than 1.0 and less than 1.6 nm. 5. Peptides according to claim 3 or 4, wherein any amino acid residue of R 1 or R 2 adjacent to an Xaa residue is neither positively nor negatively charged. 6. Peptides according to claim 3 , 4 or 5 , wherein each of Xaa 1 and Xaa 2 is, Glu and Sp is a residue of a single non-charged amino acid. 7. Peptides according to claim 3, 4, 5 or 6, wherein Sp comprises a proline residue. 8. Peptides according to any one of claims 3 to 7, wherein m and n are both 0. 9. Peptides according to any one of claims 3 to 7, wherein the sum of m plus n is 1 or 2. 10. The tripeptide Glu Pro Glu and its terminal functional derivatives . 11. Peptides according to any one of claims 3 to 10, having an N-acetyl or C-amide terminal functional group or both these groups. 12. The tripeptide N-acetyl Glu Pro Glu and its C-terminal functional derivatives.

Claims: Claims 1. Pharmaceutically acceptable compounds of less than 1200 MW for use in the treatment of IgE-mediated allergies comprising a first negatively charged atom or group and a second negatively charged atom or group, separated by a spacing group effective conformationally to position said negatively charged atoms or groups so that they will neutralize the lysine residues of the amino acid sequence Lys Thr Lys at positions 497-499 of the Ce4 constant domain of cell-bound human IgE, but excluding any compounds already known for this purpose such as DSCG and nedocromil sodium and toxic compounds. 2. Compounds according to claim 1, wherein the negative charges are separated by a distance of at least 1.0 but less than 1.6 nm. 3. Peptides of the following formula I (in which the left-hand side represents the N-terminus and the right-hand side the C- terminus) : R 1 -. Xaa 1 Sp Xaa 2 R 2 n (Formula I) wherein: R 1 represents a residue of an amino acid or of a sequence of two or three amino acids, which may be the same or different; R 2 represents a residue of an amino acid or of a sequence of two or three amino acids, which may be the same or different; (but preferably any amino acid residue of R 1 or R 2 adjacent to an Xaa residue is neither positively nor negatively charged) ; Xaa 1 represents a residue of a negatively charged amino acid, preferably Glu; Sp represents a spacing residue, preferably of a noncharged amino acid, preferably Pro, or of a non-charged dipeptide, which provides the spacing required for the negatively charged groups of the Xaa residues to be sufficiently proximal to the lysine residues of the amino acid sequence Lys Thr Lys at positions 497-499 of the Ce4 constant domain of cell-bound human IgE to neutralise them; Xaa 2 represents a residue of a negatively charged amino acid, preferably Glu; and m and n denote the number of amino acids in R 1 and R 2 respectively and each of m and n independently is 0, 1, 2 or 3 , and the sum of m plus n is from 0 to 3; and their terminal functional derivatives. 4. Peptides according to claim 3, wherein the charges on the negatively charged amino acids Xaa 1 and Xaa 2 are separated by more than 1.0 and less than 1.6 nm. 5. Peptides according to claim 3 or 4, wherein any amino acid residue of R 1 or R 2 adjacent to an Xaa residue is neither positively nor negatively charged. 6. Peptides according to claim 3, 4 or 5, wherein each of Xaa 1 and Xaa 2 is, Glu and Sp is a residue of a single non-charged amino acid. 7. Peptides according to claim 3, 4, 5 or 6, wherein Sp comprises a proline residue. 8. Peptides according to any one of claims 3 to 7, wherein m and n are both 0. 9. Peptides according to any one of claims 3 to 7, wherein the sum of m plus n is 1 or 2. 10. The tripeptide Glu Pro Glu and its terminal functional derivatives. 11. Peptides according to any one of claims 3 to 10, having an N-acetyl or C-amide terminal functional group or both these groups. 12. The tripeptide N-acetyl Glu Pro Glu and its C-terminal functional derivatives. 13. Peptides according to any one of claims 3 to 12, for use in treating an IgE-mediated allergy by prevention of triggering of histamine release by cell-bound IgE. 14. A pharmaceutical composition comprising a peptide claimed in any one of claims 3 to 12 and a pharmaceutically acceptable diluent or carrier. 15. A composition according to claim 14, in slow release form. 16. Use of a peptide claimed in any one of claims 3 to 12, for the preparation of a formulation for treating an IgE mediated allergy by prevention of triggering of histamine release by cell-bound IgE. 17. A method of treatment of IgE mediated allergy in a patient previously exposed to allergen and having allergen-specific cell-bound JgE, by prevention of triggering of histamine release by said cell-bound IgE, the method comprising administering an effective amount of a compound, peptide or composition according to any one of claims 1 to 15. 18. A method for identifying a potentially therapeutic anti- allergy small compound, which method comprises selecting a compound having all of the following three characteristics: 1) selected compound has a MW less than 1200; 2) selected compound has at least two negatively charged atoms or groups; 3) selected compound has said negatively charged atoms or groups separated by a spacing group as defined in claim 1 or 2. 19. A method for identifying a potentially therapeutic anti- allergy short peptide, which method comprises selecting a peptide having all of the following three characteristics: 1) selected peptide has six or less amino acids; 2) selected peptide has at least two negatively charged amino acid residues; 3) selected peptide has said negatively charged amino acid residues separated by a spacing group as defined in claim 3 or 4. 20. A method according to claim 18 or 19, wherein said selected compound or peptide is tested for ability to inhibit histamine release in a patient in vivo or in an in vitro model system.

Inventors: LEWIN IAN VICTOR, STANWORTH DENIS RAYMOND, LEWIN, IAN, VICTOR, STANWORTH, DENIS, RAYMOND
Applicants: LEWIN IAN VICTOR, PEPTIDE THERAPEUTICS LTD, STANWORTH DENIS RAYMOND, LEWIN, IAN, VICTOR, PEPTIDE THERAPEUTICS LIMITED, STANWORTH, DENIS, RAYMOND
Application year: 1994
Main classification: C07K5
IPC codes: A61K38, A61K49, A61P37, A61P43, C07K5, C07K7, G01N33